Clusterin pharmaceuticals and treatment methods using the same

ABSTRACT

Disclosed are pharmaceutical compositions comprising clusterin and polypeptides substantially the same as clusterin and treatment methods for inflammatory diseases and dry eye disease. The pharmaceutical compositions include an isolated clusterin or an isolated polypeptide substantially the same as clusterin. The clusterin is preferably secreted clusterin. The method of treating dry eye disease includes administering to a patient in need an effective amount of a pharmaceutical composition comprising an isolated clusterin or an isolated polypeptide substantially the same as clusterin. The method of treating a disease state characterized by inflammation includes administering to a patient having the disease state an amount of isolated clusterin or a protein substantially the same as clusterin effective to decrease the activity of a matrix metallproteinase selected from the group consisting of MMP-9, MMP-2 and MMP-7.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Non-provisionalapplication Ser. No. 12/814,349, filed Jun. 11, 2010, which claims thebenefit of the filing date of U.S. Provisional Application No.61/186,724 filed Jun. 12, 2009, all of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract Nos. R01EY12651, R01 EY09828, P30 EY14801, and P30 EY03040 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates in general to pharmaceutical compositionscomprising clusterin or polypeptides substantially the same as clusterinand to treatment methods for inflammatory diseases and dry eye disease.

BACKGROUND OF THE INVENTION

Matrix Metalloproteinases (MMPs), a family of proteolytic enzymes thatparticipate in cell migration and matrix degradation in order tomaintain and remodel the tissue structure, are zinc dependentendopeptidases. They comprise a large family of proteases that sharecommon structural and functional elements. These enzymes are primarilydistinguished from other classes of proteinases by their dependence onmetal ions and neutral pH for activity. Zymogen forms of MMPs (pro-MMPs)are secreted into the matrix of a large number of cell types. (Corbel2002) Activation of the of the pro-MMPs in the local microenvironmentcan subsequently result in discrete alterations in the tissue structure.The MMPs can be classified into distinct subclasses, two of which arethe gelatinases (MMP-2 and MMP-9) and matrilysin (MMP-7).

There has been significant interest in MMP inhibition as a therapeuticstrategy. Metal metalloproteinase inhibitors (MMPIs) have been examinedas therapeutic targets for various disease states. Animal modelsindicate that MMPIs could be useful treatments in inflammatory diseasessuch as multiple sclerosis, glomerulonephritis, bacterial meningitis,uveroentinitis, graft-versus-host disease, emphysema, aortic aneurysmand restenosis after angioplasty as a treatment for atherosclerosis.(Brinckerhoff 2002) Excessive levels of MMPs are also present in variousrespiratory diseases. Two MMPs, MMP-2 and MMP-9 have been implicated indevelopment of airway inflammation and pulmonary fibrosis and themodulation of MMP activity by eliminating excessive proteolytic damagehas been suggested. (Corbel 2001)

Tumor necrosis factor-α, TNF-α initiates most of the essential steps ofinflammation, including the increased expression of other cytokines,chemokines and proteases like MMPs. It has been demonstrated that MMP-2,MMP-9 play a role in the mouse model of TNF-induced lethal heptatitis.(See Wielockx, 2004). MMP-7 deficient mice are much less vulnerable toTNF than wild type mice, indicating MMP-7 is also important during anacute and systemic inflammation induced by TNF administration. Id. MMP 7inhibitors have been suggested for treatment of such diseases as cancer,inflammatory lung disease, Alzheimer's disease and atherosclerosis. Id.

To date however, only one MMPI, Periostat (doxycycline hydrate) islicensed in the United States. Periostat is used to treat perodontitis.Doxycline both inhibits MMP activity and also seems to decrease MMP geneexpression.

As such, there is a need for more and better agents used to treatinflammatory diseases.

Also, there is a need MMPIs capable of inhibiting or modulating MMPactivity, including the activity of MMP-2, MMP-9 and MMP-7.

MMP-9 is also implicated in diseases of the eye. MMP-9 is one of theprimary matrix-degrading enzymes on the corneal surface, and anincreased density and activity of MMP-9 was observed in the tear fluidof dry eye patients (Afonso et al., 1999; Solomon et al., 2001). Inaddition to its actions on matrix proteins, MMP-9 proteolyticallyactivates latent precursors of IL-18 and TGF-β (Schonbeck et al., 1998)and increased expression of MMP-9, IL-1b, and TNF-α have been all foundin mouse corneal epithelial cells following an experimentally inducedmodel of mouse dry eye (Solomon et al., 2001; Chen et al., 2008b). MMP-9has also been shown to be responsible for the destruction of cornealbarrier in experimental dry eye mice (Pflugfelder et al., 2005).Compared with MMP-9 deficient mice, the wild type animal sufferedgreater corneal epithelial permeability and desquamation ofdifferentiated apical corneal epithelial cells (Fini et al., 1996).

The ocular surface is covered by a tear film which is composed of threelayers: the outer oil layer, the middle aqueous layer, and the innermostmembrane/mucin layer associated with the apical corneal epithelium. Aninsufficient tear supply can cause a prolonged alteration or imbalanceof the tear film components on the surface. Such conditions alter thehomeostasis on the ocular surface by triggering stress pathways in theepithelial cells, which can lead to “dry eye syndrome (DES).” DES causesocular damage which is very painful and debilitating and can lead toblindness in severe cases. It affects tens of millions of peopleworldwide. It is accompanied by increased osmolarity of the tear filmand inflammation of the ocular surface.

In DES, the stress response made by the ocular surface epithelial cellsinitiates an inflammatory response by activating stress signal pathwaysin the epithelial cells to produce various inflammatory cytokines,chemokines, and components associated with the inflammatory process (Luoet al., 2005). The combined net result of such events is the recruitmentof inflammatory and immune cells to the ocular surface. The recruitmentof these cells induces a feed-back loop wherein the cell secretionsinduce corneal epithelial cell dysfunction and damage, which in turnleads to greater inflammation and leads to disease progression(Pflugfelder et al., 2008). Dry eye patients have increased levels ofvarious inflammatory mediators in their tear fluid such as interleukinIL-6, IL-8, and TNF-alpha, and IL-12.

To date, however, treatments for dry eye disease have been inadequate.Clusterin (CLU), also known as apolipoprotein J, testosterone-repressedprostate message-2, or sulphate glycoprotein-2, is a secreteddisulfide-linked heterodimeric glycoprotein (70-80 KD) that plays a rolein multiple biological events including apoptosis, oxidative stress,sperm maturation, complement regulation, and cytoprotection (Shannan etal., 2006). Initially, CLU was thought to be a marker of cell deathbecause its levels of expression increase in various lesions undergoingcell death. Recent studies have suggested that it also has a role inprotecting cells from cell death, and has now become a target forcertain cancer therapies (Chung et al., 2004). Following infection, theCLU levels increase as both a positive acute and chronic phase responseprotein in patients (Chen et al., 2008a) and animal models (Sharma etal., 2008).

SUMMARY OF THE INVENTION

One aspect of the present invention is the identity of novel protein andpolypeptide binding partners for MMP-2, MMP-7 and MMP-9 or the pro-MMPanalogues.

Another aspect of the present invention is the discovery that clusterinand polypeptides substantially the same as clusterin interact with andinhibit MMP-2, MMP-7 and MMP-9.

Another aspect of the present invention is the discovery that theinteraction and inhibition does not require the processing ormodification of either clusterin or the MMPs. The significance of theirinteraction was demonstrated by the observation that CLU inhibited theenzymatic activity of MMP-9.

It is another aspect of the present invention that clusterin can berendered MMP-2 and MMP-9 soluble in a non-ionic detergent condition.

Another aspect of the present invention is direct to a pharmaceuticalcomposition comprising an isolated clusterin or an isolated polypeptidesubstantially the same as clusterin. Preferably, the clusterin issecreted clusterin. Preferably, the pharmaceutical composition comprisesa carrier, and even more preferably the carrier is a sterile solution.

Another aspect of the present invention is a method of treating dry eyedisease comprising administering to a patient in need thereof aneffective amount of a pharmaceutical composition comprising an isolatedclusterin or an isolated polypeptide substantially the same asclusterin. Preferably, the pharmaceutical composition comprises secretedclusterin. Preferably, the pharmaceutical composition is administeredtopically. Even more preferably, the pharmaceutical composition furthercomprises a liquid carrier, and administration is by contacting thepharmaceutical composition to the surface of an eye of the patient.

Another aspect of the present invention is directed to a method oftreating a disease state characterized by inflammation comprisingadministering to a patient having the disease state an amount ofisolated clusterin or a protein substantially the same as clusterineffective to decrease the activity of a matrix metallproteinase selectedfrom the group consisting of MMP-9, MMP-2 and MMP-7. The disease statetreated may include inflammatory lung disease, cancer, multiplesclerosis, Alzheimer's disease, artherosclerosis, airway inflammation,pulmonary fibrosis, glomerulonephritis, bacterial meningitis,uveroentinitis, graft-versus-host disease, emphysema, aortic aneurysmand restenosis after angioplasty as a treatment for atherosclerosis.

The above-mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. The drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that CLU binds to MMP-9 in vitro, not as an enzymaticsubstrate. (A) GST-tagged MMP-9 was purified from the bacterial cellstransformed by the expression vector, and immobilized to theanti-GST-beads to prepare MMP-9-GST beads. Purified CLU-HA was incubatedwith MMP-9-GST beads (M-GST) or GST-beads (GST) in the presence(+Lysate) or absence (−Lysate) of bacterial lysate, followed by washingthe beads with binding buffer. Bound proteins were resolved ondenaturing 8% SDS/PAGE gel for the immunoblotting with anti-HA antibody.CLU cDNA detected in the yeast two hybrid assay, which corresponds tothe CLU coding sequence beginning at position 212 from the ATG site, wasused to produce a protein band of approximately 40 KDa. (B) Gelatin (20μg) and CLU (2 μg) were mixed and incubated in the presence or absenceof active MMP-9, followed by denaturing 12% SDS/PAGE gelelectrophoresis, which then was stained with Coomasie blue. Two subunitsof CLU are designated as α and β.

FIG. 2 shows MMP-9 binds to CLU in the cytoplasm of human cells. (A)pcDNA 3.1 (+) expression vectors carrying the mouse full length cDNA ofMyc-tagged CLU (CLU) or HA-tagged MMP-9 (MMP-9) were transfected, alone(CLU or MMP-9) or combined (CLU+MMP-9), into HEK293 cells. Using thewhole cell lysates of the transfected cells, immunoprecipitation (IP)with anti-HA antibody was performed to pull down MMP-9, and then theeluted samples were subjected to immunoblotting (denaturing 4-15%gradient SDS/PAGE gel) (IB) using anti-Myc antibody to detect CLU. Thebands indicated by arrows are consistent to the forms of CLU undergoingintracellular processing. in, whole cell lysate used as input; IP,immunoprecipitated sample; Unc, Uncleaved CLU protein; β,β subunit ofCLU. (B) Confocal microscopy of MMP-9 and CLU in HCLE cells. Nuclei werestained with DAPI, MMP-9 was detected by rhodamine-conjugated antibody,and CLU by FITC-conjugated antibody. In control, primary antibodies wereomitted. The images were taken at 400× magnification.

FIG. 3 shows that CLU inhibits MMP-9 and MMP-2 activities, withoutaffecting APMA-induced MMP activation. Pro-MMP-9 (A) or pro-MMP-2 (C)was incubated with PBS, CLU (50 μg/ml, or 0.63 μM), SB3-CT (2 μM), orBSA (50 μg/ml), and MMP FRET substrate peptides in the presence andabsence of 0.2 mM APMA at RT for 3 h. RFU, relative fluorescence unit,was obtained by considering the read out in PBS as 100%. Each reactionwas performed in triplicate. Standard errors were depicted as a bar ineach graph. Differences between values of PBS and CLU or SB-3CTreactions were statistically significant by Student t-test: *P=0.0004,**P=0.0001, +P=0.0003, and ++P=0.01. Gelatin zymography was performedwith the samples pooled from each reaction set. Pro-MMPs, processed MMPs(MMP-9 or MMP-2), and C-terminal truncated, processed MMP-9 (MMP-9-ΔC)were indicated by arrows. (B) Dose-dependent inhibition of MMP-9 by CLUor SB-3CT was performed in the same condition.

FIG. 4 shows that inhibition of MMP-9 by CLU does not require theC-terminal hemopexin-like domain. Recombinant MMP-9 catalytic domain (A)or APMA-activated MMP-7 (B) was incubated with PBS, CLU (50 μg/ml or0.63 μM), SB3-CT (2 μM), or BSA (50 μg/ml), and MMP FRET substrate at RTfor 3 h. RFU in PBS samples was considered 100%. Each reaction wasperformed in triplicate. Standard errors were depicted as a bar in eachgraph.

FIG. 5 shows that CLU enhances the solubility of MMP-9 and MMP-2. (A)Pro-MMP-2 or pro-MMP-9 was incubated in the buffer containing 0.04% ofBrij 35, NP-40, or N-octyl glucopyranoside (OG) in the presence of CLU(C) or PBS (P), and then the soluble and insoluble fractions wereobtained by centrifugation to resolve on the gelatin zymography.Relative soluble fraction was calculated by dividing the soluble densitywith total density of each set, obtained using Image J analysis. (B)Pro-MMP-2, (C) Pro-MMP-9 or APMA-processed active MMP-9 inhibitionassays were performed in the reaction buffer containing 0.04% of Brij 35or NP-40.

FIG. 6 shows that Pro-MMP-9 binds with higher affinity to CLU thanprocessed MMP-9. (A) Recombinant proteins, Pro-MMP-9, MMP-9, MMP-9-ΔC,CLU-His were incubated together, and bound to agarose beads coupled oruncoupled with Anti-(His)₆ antibody in two different binding bufferscontaining Brij 35 concentrations, 0.04% or 0.002%. MMPs bound to thebeads were obtained by washing with the corresponding binding buffer toresolve on gelatin zymography. Three different volumes of each samplewere loaded to compare the relative density. (B) Pro-MMP-9 bound to CLUcoupled to agarose beads, prepared as in (A), was incubated in thepresence or absence of APMA for 1 h at RT, prior to separation of beads(B) and supernatant (S) fractions, which were resolved by zymography.Arrow indicates MMP-9 processed by APMA.

FIG. 7 shows that CLU binds to MMP-2 and MMP-9. A mixture of MMP-2,MMP-9, and C-terminal truncated MMP-9 (9-ΔC) was incubated withHis-tagged CLU, used in a pull-down assay and resolved on a gelatinzymography gel. The input and bound samples were loaded in a series ofdilution.

FIG. 8 shows that CLU is expressed and secreted from corneal epithelialcells. (A) Gel of isoforms 1, 2, and 3 of total RNA purified from HCLEcells (I), human corneal epithelial tissue (II), and human periodontalligament stem cells (III). A reverse transcriptase minus (−RT) samplewas used as a control.

The arrow head indicates a DNA marker of 400 bp. (B) Western blot of themedia from the HCLE cell culture with anti-CLU antibody sc-6419. Lane 1contains conditioned medium (K-sfm) from the cell culture; lane 2 hasregular medium (K-sfm plus BPE and EGF) from the culture; and lane 3includes fresh regular medium as a control. The arrow indicates theposition of the β-subunit of CLU with the expected size of ˜40 KD.

FIG. 9 shows that intracellular processing of CLU is affected by growthconditions. Western blot using anti-CLU antibody of HCLE cells grown atthe subconfluent cell density (Sub-C), confluent cell density (C), orstratified corneal cells (S). The arrows indicate CLU bands thatincreased or decreased in the stratified cells, as compared to the othertwo.

FIG. 10 shows that Clusterin inhibits the induction of MMP-9 byTNF-alpha from human corneal limbal epithelial (HCLE) cells. HCLE cellswere grown to be confluent in KSFM media and then replenished withDMAM/F12 media to stratify cells for 7 days. To these cells inserum-free DMEM/F12 media, clusterin (50 ug/ml), bovine serum albumin(BSA, 50 ug/ml), and TNF-alpha (10 ng/ml), individually or incombination, were treated for 24 hours. The same volume of supernatantsof the cell cultures treated were collected to resolve on SDS/PAGE gelcontaining gelatin in order to perform gelatin zymography to visualizethe presence of MMP-9 secreted from the cells.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

MMP, matrix metalloproteinase;

CLU, clusterin;

TIMP, tissue inhibitor of matrix metalloproteinase;

BSA, bovine serum albumin;

APMA, 4-aminophenylmercuric acetate;

SB-3CT, 3-(4-phenoxyphenylsulfonyl)-propylthiirane;

FRET, fluorescence resonance energy transfer

Definitions

Unless otherwise indicated, all terms used herein have the meaningsgiven below, and are generally consistent with same meaning that theterms have to those skilled in the art of the present invention. It isto be understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

The term “clusterin” refers to human clusterin, including secretedclusterin and nuclear clusterin, or any subunit, fragment or region ofeither capable of binding to, or reducing the activities of MMP-9, MMP-2or MMP-7. The term clusterin optionally encompasses non-peptidiccomponents, such as carbohydrate groups or any other non-peptidicsubstituents that may be added to clusterin by a cell in which theprotein is produced, and may vary with the type of cell.

The terms “treatment” or “treating” refers to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) the targeted pathologic condition ordisorder. It may also encompass relief of symptoms associated with apathological condition or disorder. Those in need of treatment includethose already with the disorder as well as those prone to have thedisorder or those in whom the disorder is to be prevented.

An “effective amount” of isolated clusterin or an isolated polypeptidesubstantially the same as clusterin is an amount sufficient to decreasethe activity or the amount of a target matrix metalloproteinase. An“effective amount” may be determined empirically and in a routinemanners in relation to the stated purpose.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS9™.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “substantially the same” refers to nucleic acid or amino acidsequences having sequence variation that do not materially affect theability of the amino acid sequence to bind to or reduce the activity ofthe target metalloproteinase, e.g. MMP-9, MMP-7, MMP-2. With particularreference to nucleic acid sequences, the term “substantially the same”is intended to refer to the coding region and to conserved sequencesgoverning expression, and refers primarily to degenerate codons encodingthe same amino acid, or alternate codons encoding conservativesubstitute amino acids in the encoded polypeptide. With reference toamino acid sequences, the term “substantially the same” refers generallyto conservative substitutions and/or variations in regions of thepolypeptide not involved in determination of structure or function.

As used herein, the term “expression” or “gene expression” refers to theprocess of converting genetic information encoded in a gene into RNA(e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene(i.e., via the enzymatic action of an RNA polymerase), and for proteinencoding genes, into protein through “translation” of mRNA. Geneexpression can be regulated at many stages in the process.“Up-regulation” or “activation” refers to regulation that increases theproduction of gene expression products (i.e., RNA or protein), while“down-regulation” or “repression” refers to regulation that decreaseproduction. Molecules (e.g., transcription factors) that are involved inup-regulation or down-regulation are often called “activators” and“repressors,” respectively. A protein is “inappropriately expressed” ifit is expressed in different places, at different times, or in differentamounts in an organism characterized by a disease state than it is in anorganism with no disease.

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

Pharmaceutical Compositions

One aspect of the present invention is directed to a pharmaceuticalcomposition comprising an isolated clusterin or an isolated polypeptidesubstantially the same as clusterin. Preferably, the clusterin issecreted clusterin. Preferably, the pharmaceutical composition comprisesa carrier, and even more preferably the carrier is a sterile solution.

Human clusterin (CLU) is composed of two disulfide-linked α (34-36 kD)and β (36-39 kD) subunits derived from a single amino acid chain (449amino acids in human) that becomes glycosylated in the endoplasmicreticulum and Golgi bodies and undergoes intramolecular cleavage anddimerization before secretion. The first 22 amino acids comprise thesecretory signal sequence. The cleavage site between the α and β chainsis between amino acids 227 and 228. Clusterin contains three hydrophobicdomains, a long α-helix motif near the amino terminal and at least sixN-linked glycosylation sites. Clusterin also contains a hemopexin-likedomain at the C-terminus of the enzyme, which modulates the processingand activity of the enzymes by serving as a binding region forregulatory or target proteins

The sequence listing of Clusterin Isoform 2 Preproprotein [Homo sapiens](SEQ ID NO: 1) (NCBI Reference Sequence: NP_(—)976084.1) is as follows:

ORIGIN 1 mmktlllfvg llltwesgqv lgdqtvsdne lqemsnqgskyvnkeiqnav ngvkqiktli 61 ektneerktl lsnleeakkk kedalnetre setklkelpgvcnetmmalw eeckpclkqt 121 cmkfyarvcr sgsglvgrql eeflnqsspf yfwmngdrid sllendrqqt hmldvmqdhf 181 srassiidel fqdrfftrep qdtyhylpfs lphrrphfff pksrivrslm pfspyeplnf 241 hamfqpflem iheaqqamdi hfhspafqhp ptefiregdd drtvcreirh nstgclrmkd 301 qcdkcreils vdcstnnpsq aklrreldes lqvaerltrk ynellksyqw kmlntsslle 361 qlneqfnwvs rlanltqged qyylrvttva shtsdsdvps gvtevvvklf dsdpitvtvp 421 vevsrknpkf metvaekalq eyrkkhree

In vivo, the human precursor polypeptide chain is cleavedproteolytically to remove the 22 amino acid secretory signal peptide andsubsequently between residues 227/228 to generate the alpha and betachains. These are assembled in an anti-parallel fashion to give aheterodimeric molecule in which the cysteine-rich centers are linked byfive disulfide bridges and are flanked by two predicted coiled-coilalpha-helices and three predicted amphipathic alpha-helices.

The clusterin of the present invention can be human clusterin, includingsecreted clusterin and/or nuclear clusterin, or any subunit, fragment orregion of either capable of binding to, or reducing the activities ofMMP-9, MMP-2 or MMP-7. The subunits, fragments or regions may be testedfor ability to bind or reduce the activities of MMP-9-MMP2 or MMP-7 asdescribed in the accompanying Examples. Acceptable subunits may includehuman or secreted clusterin without the secretary signal sequence and/orwithout the hemopexin-like domain at the C-terminus of the enzyme. Theterm clusterin also encompasses polypeptides with optional non-peptidiccomponents, such as carbohydrate groups or any other non-peptidicsubstituents that may be added to clusterin by a cell in which theprotein is produced, and may vary with the type of cell.

Recombinant human clusterin may be purchased from any number of knownsources, expressed in cell lines of mouse and human. It may also beisolated from human serum by known methods. Any subunit, fragment orregion may be isolated or synthesized according to known techniques forpolypeptide synthesis.

The pharmaceutical compositions of the present invention may alsoinclude polypeptides substantially the same as human clusterin, secretedclusterin, nuclear clusterin or any subunit, fragment or region ofeither capable of binding to, or reducing the activities of MMP-9, MMP-2or MMP-7. Generally, amino acid sequences are substantially the same ifthey have a sequence variation that do not materially affect the abilityof the protein, subunit, fragment or region bind to or reduce theactivity of MMP-9, MMP-2 or MMP-7. These polypeptides can contain, forexample, conservative substitution mutations, i.e. the substitution ofone or more amino acids by similar amino acids. For example,conservative substitution refers to the substitution of an amino acidwith another within the same general class such as, for example, oneacidic amino acid with another acidic amino acid, one basic amino acidwith another basic amino acid or one neutral amino acid by anotherneutral amino acid. What is intended by a conservative amino acidsubstitution is well known in the art. The subunits, fragments orregions may be tested for ability to bind or reduce the activities ofMMP-9-MMP2 or MMP-7 as described in the accompanying Examples. Thepolypeptides of the present invention may be made by known techniquesfor polypeptide synthesis.

The polypeptides of the present invention which occur naturally, or aresynthesized according to known methods, are generally “isolated.”Specifically, the should be used in the pharmaceutical composition ofthe present invention in a condition other than their respective nativeenvironment, such as apart from blood and animal tissue. In a preferredembodiment, the isolated polypeptide is substantially free of otherpolypeptides, particularly other polypeptides of animal origin. It ispreferred to provide the polypeptides in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders can be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutionsthat can also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, and drops. The compositions of the present invention can alsobe formulated as suspensions in aqueous, non-aqueous or mixed media.Aqueous suspensions can further contain substances that increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension can alsocontain stabilizers.

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions can contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or cancontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

Dosing is also dependent on severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until symptomatic relief or a cure iseffected or a diminution of the disease state is achieved. Theadministering physician can determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages can vary dependingon the relative potency of individual polypeptide and should generallybe sufficient to reduce the activity of the target MMP, either MPP-2,MMP-9 or MMP-7. Following successful treatment, it can be desirable tohave the subject undergo maintenance therapy to prevent the recurrenceof the disease state, wherein the polypeptide is administered inmaintenance doses.

An especially preferred dosage form is a sterile solution for topicaluse, such as use as drops. Therapeutic formulations are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable liquid carrier, andoptionally other excipients or stabilizers (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)), to produce an aqueoussolution or suspension. Acceptable carriers, excipients or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, ordextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol.

The solution or suspension formulations should be sterile. This isreadily accomplished by filtration through sterile filtration membranes,prior to or following lyophilization and reconstitution. The resultingtherapeutic compositions herein generally are placed into a containerand the route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Treatment Methods for Dry Eye Disease

Another aspect of the present invention is a method of treating dry eyedisease comprising administering to a patient in need thereof aneffective amount of a pharmaceutical composition comprising an isolatedclusterin or an isolated polypeptide substantially the same asclusterin.

It should be understood that the terms “treatment” or “treating” refersto both therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) the targetedpathologic condition or disorder. Treatment should also be understood toinclude relief of the symptoms of the disease. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

An “effective amount” of isolated clusterin or an isolated polypeptidesubstantially the same as clusterin is an amount sufficient to decreasethe activity or the amount of a target matrix metalloproteinase. In thecase of dry eye disease, the target metalloproteinase is preferablyMMP-9. Generally, not all the activity target metalloproteinase need beeliminated. Rather, the activity of the target metalloproteinaise needonly be reduced by an amount sufficient to reach the therapeutic goal.For instance, if the goal of the treatment is preventative, the amountof the reduction of activity need only be sufficient to prevent dry eyedisease. Thus, an effective amount may be determined empirically and ina routine manner in relation to the stated purpose.

The clusterin used in the connection with the method may be any isolatedclustering or a polypeptide substantially the same as clusterin that canreduce the activity of the target metalloproteinase in the eye. Mostpreferably, the pharmaceutical composition comprises secreted clusterin.

In accordance with the present invention, clusterin can inhibit theactivity of MMP-9 extracellularly in epithelial cells. As such, thepharmaceutical composition for use in the treatment of dry eye diseaseis administered topically. Preferably, when topically administered, theclusterin is combined with a liquid carrier, and administration in theform of an eye drop by contacting the pharmaceutical composition to thesurface of an eye of the patient. The concentration of the clusterin inthe resulting liquid pharmaceutical should be sufficiently high thatwhen administered to the patient, there is a sufficient concentration ofclusterin to reduce the activity sufficient to produce the requiredresult.

Treatment Method for Inflammatory Diseases

Another aspect of the present invention is directed to a method oftreating a disease state characterized by inflammation comprisingadministering to a patient having the disease state an amount ofisolated clusterin or a protein substantially the same as clusterineffective to decrease the activity of a matrix metallproteinase selectedfrom the group consisting of MMP-9, MMP-2 and MMP-7. The disease statetreated may include inflammatory lung disease, cancer, multiplesclerosis, Alzheimer's disease, artherosclerosis, airway inflammation,pulmonary fibrosis, glomerulonephritis, bacterial meningitis,uveroentinitis, graft-versus-host disease, emphysema, aortic aneurysmand restenosis after angioplasty as a treatment for atherosclerosis.

It should be understood that the terms “treatment” or “treating” refersto both therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) the targetedpathologic condition or disorder. Treatment should also be understood toinclude relief of the symptoms of the disease: Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

An “effective amount” of isolated clusterin or an isolated polypeptidesubstantially the same as clusterin is an amount sufficient to decreasethe activity or the amount of a target matrix metalloproteinase. Theidentity of target metalloproteinase, either MMP-9, MMP-7 or MMP-2, willgenerally depend on the disease state to be treated. Generally, not allthe activity target metalloproteinase need be eliminated. Rather, theactivity of the target metalloproteinaise need only be reduced by anamount sufficient to reach the therapeutic goal. In addition, sincenormal functioning of tissue associated with the inflammation mayrequire some level of target metalloproteinase activity, the activity ofthe target metalloproteinase should not be reduced below a level neededfor adequate functioning of the target tissue. Thus, an effective amountmay be determined empirically and in a routine manner in relation to thestated purpose. For instance, if the goal of the treatment ispreventative, the amount of the reduction of activity need only besufficient to prevent dry eye disease.

The clusterin used in the connection with, the method may be anyisolated clusterin or a polypeptide substantially the same as clusterinthat can reduce the activity of the target metalloproteinase in thetissue affected by the disease state.

Administration can be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof. While suchexamples are typical of those that might be used, other procedures knownto those skilled in the art may alternatively be utilized. Indeed, thoseof ordinary skill in the art can readily envision and produce furtherembodiments, based on the teachings herein, without undueexperimentation.

Materials and Methods

Cells—Cells of the human embryonic kidney cell line, HEK293 were grownin DMEM containing 10% FBS, 100 units/ml penicillin, and 100 μg/mlstreptomycin in a cell culture incubator at 37° C., 5% CO₂. Immortalizedhuman corneal-limbal epithelial (HCLE) cells, kindly provided by Dr.Ilene Gipson (Harvard University) were grown in Gibco keratinocyte SFMmedia (Gibco BRL, Carlsbad, Calif.) [Argueso, 2006].

MMP-9 cDNA Constructs—The entire MMP-9 cDNA (coding amino acid 1-703)was reverse transcribed and amplified from mouse muscle tissue.

(SEQ ID NO: 2) Primers MMP9.1 (5′-GGCGCCGAATTCATGAGTCCCTGGCAGCCCCTG-3′)and (SEQ ID NO: 3) MMP9.2109(5′-GGGCCCGTCGACTCAAGGGCACTGCAGGAGGTCGTAGGTCA-3′)incorporating EcoR I and Sal I sites, respectively, was used. Thisrt-PCR product was inserted into the yeast expression vector pGBKT7 andlabeled pGB-M9. Utilizing the pGB-M9 plasmid as a template an N-terminusMMP-9truncated PCR product missing the signal and propeptide domain(amino acid 109-703) was generated using primers MMP-9.325(5′-GGCGCCGAATTCCAAACCTTCAAAGGCCTCAAGTGGG-3′) (SEQ ID NO: 4)-MMP9.2109primers and labeled pGB-M9ΔPP. The GST tagged MMP-9 constructs weregenerated by ligation of the pGB-M9 and pGB-M9ΔPP EcoR I/Sal I insertsinto the pGEX 4T vector (Amersham, Piscataway, N.J.).

Library Construction and Screening—Whole mouse cornea was excised andtotal RNA isolated via the TRIzol reageant (Invitrogen, Carlsbad,Calif.). The cDNA library was synthesized using 2 μg total RNA andrandom primers per the protocol for the Matchmaker Two-Hybrid System(Clontech, Mountain View, Calif.). This yeast two hybrid system utilizesan in vivo library construction method; therefore the cDNA library andthe linearized yeast expression vector pADT7-Rec were simultaneouslytransfected into the AH109 pGB-M9ΔPP stable yeast cells. Positivecolonies were selected on quadruple knock-out plates, yeast minimal agarplates lacking tryptophan, leucine, histidine and adenine. The tworeporter genes testing for interactions are the histidine and adenine.An additional screen involved the addition of X-α-galactosidase to thequadruple knocked-out media, whereby a positive interaction allowed forblue/white screening.

Yeast Plasmid Isolation—Positive colonies were inoculated into anovernight culture of YAPD media and plasmid isolation performed uponsaturation using the following yeast lysis method. Briefly, pelletedyeast cells were vortexed in equal volume yeast lysis buffer (2% TritonX-100, 1% SDS, 0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) andphenol-chloroform-isoamyl alcohol [25:24:1] with acid washed glassbeads. The clarified supernatant was ethanol precipitated in thepresence of NaOAc. The resulting DNA complex was transformed intoXL1-blue bacterial cells and colonies selected on ampicillin plates toselect for the pADT7-library clone. The resulting bacterial colonieswere lysed via the standard alakaline lysis protocol and the plasmidssequenced.

Sequence analysis—The DNA sequence were translated based on the readingframe given for the pADT7-Rec vector and both the protein sequence andDNA sequence subjected to BLAST searches in GENBANK. The DNA sequencewere translated into protein sequence using the translate tool at theExPASy Bioinformatics Resource Portal website. Dual searches were doneto confirm that the protein sequence translated matched their DNAsequences, mismatching clones were discarded.

GST Pull-Down Assays—The GST tagged MMP-9 constructs were generated byligation of the pGB-MMP-9 EcoR I/Sal I inserts into the pGEX 4T vector(Amersham, Piscataway, N.J.). The pGEX 4T MMP-9 constructs weretransformed into the E. Coli strain BL21(DE3) (Novagen, Gibbstown, N.J.)for expression. The bacterial pellet was prepared from a 100 ml culturethat was grown to mid-log phase, induced with 0.4 mM isopropyl β-D-thiogalactopyranoside for three hours, followed by centrifugation. Thepellet was resuspended and lysed by sonication in ST buffer (50 mM TrispH 7.4, 100 mM NaCl, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF, 1mM NaF), and GST-MMP-9 protein purified via incubation with glutathionecoated beads (Amersham, Piscataway, N.J.) overnight at 4° C. The nextday the beads were extensively washed in ST buffer. To confirmproduction of GST-MMP-9 protein, the beads were electrophoresed on an 8%SDS-PAGE gel. The gel was subjected to Coomassie Brilliant Blue Stainingor transferred to PVDF membrane and immunoblotted using a rabbitpolyclonal antibody to MMP-9 (Triple Points Biologics, Inc.). Using theTNT quick coupled transcription/translation system (Promega, Madison,Wis.) the pADT7-CLU clone generated an HA tagged CLU fusion protein. TheGST-MMP-9 or GST coated beads were incubated with equal amounts ofHA-CLU overnight at 4.0 in immunoprecipitation (IP) buffer (50 mM Tris,pH 7.4, 100 mM NaCl, 0.1% Triton X-100, 1 μg/ml leupeptin, 1 μg/mlaprotinin, 1 mM PMSF, 1 mM NaF) in the presence or absence of 1 mg/mlBL21 bacterial soluble extract (to decrease non-specific background).The following day the beads were washed 3 times in IP buffer,resuspended in SDS-PAGE loading buffer, electrophoresed on an 8%SDS-PAGE gel, transferred and immunoblott using a mouse HA antibody(Santa Cruz, Calif.).

Generation of expression vectors for MMP-9 and CLU- The pcDNA3.1(+)expression vector (Invitrogen, Carlsbad, Calif.) was used to insert PCRfragment generated using pGB-M9 as a template with the gene-specificprimers containing EcoR I (in forward primers) or Not I (in reverseprimers) sites: Mouse CLU with C-terminal a Myc tag (CLU-Myc),5-GATCGAATTCATGAAGATTCTCCTGCTGT (SEQ ID NO: 5) and5′-CGATGCGGCCGCTCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCTTCCGCACGGCTTTTCCT (SEQID NO: 6) ; and mouse MMP-9 with a C-terminal HA tag (MMP-9-HA), 5′-GATCGAATTCATGAGTCCCTGGCAGCC (SEQ ID NO: 7) and5′-CGATGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTAAGGGCACTGCAGGAGGT (SEQ IDNO: 8). The DNA sequence of cloned DNA was confirmed by DNA sequencing.

Immunoprecipitation Assay—HEK293 cells were grown in subconfluentculture in 6-well plate, and transfected with 2.5 μg of pcDNA3.1(+)/MMP-9, pcDNA3.1 (+)/MMP CLU, or both, using Lipofectamine LTXaccording to the manufacturer's instruction (Invitrogen Inc., Carlsbad,Calif.). One day post transfection, whole cell extracts were preparedusing RIPA buffer, incubated with anti-HA antibody to immunoprecipitateHA tagged MMP-9 and partners. The complexes were resolved on adenaturing SDS/PAGE gel for immunoblotting to detect Myc tagged CLUproteins with anti-Myc antibody.

Confocal Microscopy—HCLE (5000 cells/well) were plated into 16-wellchamber slide. The cells were allowed to adhere and proliferate for 48hours before immunocytochemical analysis. The cells were washed oncewith PBS and then fixed in ice cold methanol for 10 minutes. Next, thecells were washed three times with PBS followed by blocking with 1% BSAand 0.25% Triton-X 100 in PBS for one hour at room temperature. Thecells were then incubated at room temperature for 45 minutes with bothgoat anti-CLU (1:50) and rabbit anti-MMP-9 (1:50) antibodies in theblocking solution. Following the primary antibody incubation, cells werewashed three times with PBS for 5 minutes each then incubated at roomtemperature for one hour with both donkey anti-goat-FITC (1:2000) andgoat anti-rabbit-Rhodamine (1:2000) secondary antibodies. Subsequently,the cells were washed three times with PBS for five minutes each andrinsed once with distilled water. The cells were then covered with adrop of vectastain (containing DAPI) and coverslipped. Cells were imagedon a Perkin Elmer Ultraview ERS Spinning Disk Laser Confocal Microscopeat a total magnification of 400 times.

MMP inhibition assay—MMPs were mixed with CLD in TSCB buffer, 50 mMTris-HCI, pH, 7.5, 150 mM NaCl, 5 mM CaC₂, 0.002% Brij35 (or otherwiseindicated concentration) 30 min prior to substrate addition, andincubated for 3 h at RT in the presence or absence of 0.02 mMp-aminophenylmercuric acetate (APMA). MMP activity was measured using a5-FAM/QXL520 fluorescence resonance energy transfer (FRET) peptide as asubstrate (EXlEm=490 nm/520 nm) (AnaSpec, Fremont, Calif.), according tothe manufacturer's protocol. Relative fluorescence unit (RFU) wasobtained by subtracting the values of APMA- or enzyme-omitted reactionsfrom those of test samples, and then considering the fluorescence unitof a PBS (vehicle) sample as 100%. Enzyme reactions were performed inblack 96-well plate, containing combinations of 10 ng, 5 ng, 5 ng, 10ng, 2.5 μg, 2.5 μg of MMP-2, MMP-7, catalytic domain of MMP-9, MMP-9,CCLU, and/or BSA, respectively, in 50 μl reaction volume. As MMPsubstrates (0.4 μM), 520 MMP FRET substrate I,QLX520-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-Lys(5-FAM)-NH₂ (SEQ ID NO: 9), wasused for MMP-2 and MMP-9, and 520 MMP FRET substrate XIV,QXL520-γ-Abu-Pro-Cha-Abu-Smc-His-Ala-Dab(5-FAM)-Ala-Lys-NH₂ (SEQ ID NO:10), used for MMP-7 reactions, both of which were purchased from AnaSpec(Fremont, Calif.).Recombinant proteins such as mouse CLU with C-terminal(His)₆ (CLU-His), MMP-2, and MMP-7 were purchased from R&D Systems(Minneapolis, Minn.), MMP-9 was purchase from Ana-Spec, Inc. (San Jose,Calif.). The recombinant catalytic domain of human MMP-9 (residue112-445) was purchased from ProtEra (Sesto Fiorentino, Italy). Agelatinase-specific synthetic inhibitor, SB-3CT, was obtained fromBiomol International (Plymouth Meeting, Pa.). Fluorescence was monitoredusing a Victor³™ V multilabel counter (PerkinElmer). For the gelatindigestion, a 50 μl mixture of gelatin (20 μg) and CLU (2 μg) wasincubated with or without active MMP-9 (70 ng) for 2 hours at 37° C. Thereaction products were run on a 12% denaturing SDS/PAGE gel for Coomasiestaining.

Gelatin Zymography—Zymographic analysis was used to analyze the relativeamounts of MMPs and was performed according to the procedure describedby [Gordon, 2009]. Briefly, Samples were loaded into an 8% PAGE gelcontaining 0.1% gelatin (Sigma-Aldrich). Gels were run for 45 min at 200V. Gels were then incubated in 2.5% Triton-X for 1 h at RT on a rocker.Gels were thoroughly washed with distilled water and incubated overnightin renaturing buffer (10 mM CaCl₂, 50 mM Tris-HCl pH 7.5) at 37° C. Thenext morning, gels were briefly washed and then stained (25 g Coomassieblue, 150 ml isopropanol, 50 ml acetic acid, 300 ml H2O) for 1 h at RTon a rocker. Gels were then de-stained in distilled water until bandswere clearly visible. Gels were then scanned for densitometric analysis.

Gelatinase Precipitation Assay—pro-MMP-2 or -9 (5 ng/tube) wereincubated in TSCB buffer containing 0.05% non-ionic detergents indicatedin the presence of CLU (1.25 μg/tube) or PBS (vehicle control) at RT for80 min, followed by microcentrifugation for 2 min at 15K RPM to separatethe soluble and insoluble fractions, which were resolved in zymography.Densities of the bands were quantified using Image J analysis. Relativesolubility was calculated by dividing the density of soluble band withthe combined densities of soluble and insoluble bands in each set ofreactions.

In Vitro Protein Binding Assay—To make various processed forms of MMPs,pro-MMPs were incubated with 1 mM APMA in TSCB buffer at 37° C. for 4 hto make C-terminal truncated processed MMP-9 (MMP-9-ΔC) [Murphy, 1995].CLU-His (2.5 μg) was incubated with mixture of 30-100 ng of pro-MMP-9,MMP-9, and MMP-9-ΔC, in the TSCB buffer with 0.002 or 0.04% Brij 35 for1 h at 4° C. followed by additional incubation for 1.5 h with anti-Histag antibody-conjugated agarose beads. The beads were washed three timeswith the binding buffer, and dissolved in 1×SDS sample loading buffer.Relative amounts of bound MMPs were assessed on the gelatin zymography.In order to perform CLU binding domain analysis, pro-MMP-9 bound toCLU-coupled beads was prepared, as described above, and then dividedinto two aliquots. They were incubated with and without 1 mM APMA at RTfor 1 h in TSCB buffer on the rotator. The beads (B) were spun down andsolution (S) was separated. The beads were washed with TSCB andresuspended in SDS sample loading buffer. Equivalent volume of S and Bfraction were subjected to gelatin zymography.

Example 1 Interaction of CLU with MMP-9

Previous results indicate that CLU protein can bind to MMP-9 withoutundergoing post-translational processing of the protein. To examine thepossibility that they can form a complex inside the cells, weconstructed two expression vectors carrying mouse cDNA of Myc-tagged CLUor HA-tagged MMP-9 protein. We transfected HEK293 cells with the twovectors, alone or combined, and then performed the immunoprecipitation(IP) assay using the whole cell lysates of the transfected cells to pulldown MMP-9, and then used Western blotting to detect any CLU proteinpulled down together with MMP-9 (FIG. 2A).

The bands indicated by arrows (FIG. 2A) are consistent with the forms ofCLU undergoing intracellular processing (Burkey et al., 1991). MultipleCLU bands were detected in the IP sample using the cotransfected celllysate that were not detected in the IP from CLU transfection alone,indicating that they form a complex with MMP-9 inside the cells. Theseresults suggest the possibility that MMP-9 keeps CLU inside the cells byinteracting with those proteins during processing. As such, CLUintermediates were not detected without MMP-9 coexpression (de Silva etal., 1990; Burkey et al., 1991). This may support and explain a previousreport that shows that processing and secretion takes place veryquickly, with a half-time of 30-35 min (Burkey et al., 1991).

Next, we tested the interaction of secreted CLU using mouse recombinantproteins. We used purified recombinant proteins of MMP-2, MMP-9, and CLUthat were secreted from a mouse myeloma cell line (NSO). All of theproteins were purchased from R&D Systems. A mixture of MMP-2, MMP-9, andC-terminal truncated MMP-9 (9-ΔC) was incubated with His-tagged CLU, andsubjected to the pull-down assay, using 150 mM NaCl-containing bindingbuffer and anti-His tag antibody resins. Portions of the input samples(Input) and proteins bound to the antibody (Bound) were resolved on thegelatin zymography gel.

The recombinant CLU contains a (His)₆-Tag at the C-terminus of theprotein, which was used for pull-down experiments with anti-His tagantibody beads. It was previously reported that CLU also binds toMT6-MMP/mmp-25 and that the hemopexin domain in the MMP C-terminalregion is required for their interaction (Matsuda et al., 2003). To useas an internal control for the binding experiment, we removed part ofMMP-9 (MMP-9-ΔC) by promoting self-processing by APMA(4-aminophenylmercuric acetate) treatment. For the in vitro bindingassay we mixed MMP-2, MMP-9, and MMP-9-ΔC (FIG. 7).

The initial MMP-9 protein shows a high molecular weight smear aboveMMP-9, which are presumably aggregates of MMP-9, since we did not detectthem in the heat treated reducing condition used for Western blotting(FIG. 8). The input and bound samples were loaded in a series ofdilution so that the relative densities of the bands could be easilyassessed.

After comparing the relative densities of protein bands in the same lanefor the input and the bound, we found that MMP-9 was highly enriched inthe bound sample. Though weaker than MMP-9, MMP-2 also had an affinityfor CLU while the truncated MMP-9 did not. We conclude that MMP-2 andMMP-9 interact with CLU.

Example 2 CLU and MMP-9 Interact Inside and Outside of Cells

To identify corneal proteins, including CLU, that interact with MMP-9,the yeast two hybrid system was employed. We used mouse MMP-9 cDNAcorresponding to active MMP-9 lacking the N-terminal signal peptide andpropeptide domains. Recovery of the colonies supported on the knock outmedia and expressing galactosidase activity identified 24 potential preysequences. Sequence analysis of the rescued pADT7 plasmids found manycandidates known to be present in extracellular region or in cellmembrane. Many candidates were also observed to contain zinc fingerdomains, coiled-coiled domains, or loop-helix-loop domains, well-knownregions for protein-protein interactions. One of the sequencesidentified was CLU. The CLU clone that was isolated from the yeast assaycontained cDNA beginning at position 212 from the ATG site, thus lackingN-terminal 71 amino acids.

To further confirm the interaction between MMP-9 and CLU that wasdetected by yeast two hybrid screening, we performed GST pull down assayusing recombinant GST-tagged MMP-9 fusion protein. The GST-MMP-9 wasimmobilized on a glutathione-agarose matrix and incubated with in vitrotranslated HA-tagged CLU (FIG. 1A) in the presence or absence of solublebacterial lysate as a nonspecific competitor. Our results showed thatMMP-9 and CLU still interacted even in the presence of the bacteriallysate, confirming the results from the yeast two hybrid system.

Next, as there are various known MMP-9 substrates, we tested apossibility that CLU might be a novel enzymatic substrate of MMP-9. Tothis end, in a preliminary experiment, we digested CLU with active MMP-9to find that CLU was not digested by MMP-9. To confirm this, both CLUand gelatin, a known substrate for MMP-9, were incubated in the presenceor absence of an excessive amount of active MMP-9, and then resolved onthe denaturing SDS/PAGE gel followed by Coomasie staining (FIG. 1B). Asshown on the gel, the density of CLU protein, separated as α and βsubunits, changed little, whereas most high molecular weight gelatindisappeared upon incubation with MMP-9, indicating that CLU is not asubstrate for MMP-9 in vitro.

Next, we tested whether MMP-9/CLU interaction also occurs in vivo. UsingHEK293 cells, a human embryonic kidney cell line, mouse HA-tagged MMP-9and Myc-tagged CLU expression vectors were transfected individually orin combination (FIG. 2A). Our results showed two protein bandscorresponding to the full length unprocessed glycosylated form (˜60 kDa)of CLU as well as processed CLU β-subunit (˜37 kDa) uponimmunoprecipitation (IP) with anti-HA antibody followed byimmunoblotting with anti-Myc antibody when both CLU and MMP-9′ wereexpressed together. CLU exists in the cytoplasm as ˜50 kDa and ˜60 KDaprecursor forms (depending on the extent of glycosylation) of thesecretory CLU [Trougakos, 2009]. We also recognized that a stronger bandof ˜50 KDa, compared with the other two IP lanes, was detected inco-transfected IP lane, which might be a non-glycosylated full-lengthform of CLU. Unfortunately, this band is overlapped with IgG heavy chainband from the antibodies used for IP. These results suggest that MMP-9and CLU interact in vivo even before processing of the proteins.However, we failed to observe coimmunoprecipitation at the endogenouslevel using the HOLE whole cell extract; at present, we ascribed thisfailure to two possibilities, that antibodies used may interfere withand dissociate the interaction between of the two proteins, and/or thatendogenous level of both proteins might not be enough to performimmunoprecipitation as both proteins are secreted immediately aftertheir synthesis. As an alternative way to confirm their interaction invivo, we localized the two proteins using confocal microscopy (FIG. 2B).In control without primary antibody incubation, neither MMP-9 nor CLUwas localized. However, with specific antibodies both proteins werefound to be present surrounding the nuclei, and the composite image ofthe two proteins indicated that both proteins are co-localized, probablyin the secretory pathway (ER and/or Golgi compartment). The combinedresults of the in vitro binding assay, the IP, and confocal analysissuggest CLU and MMP-9 interact inside and outside cells.

Example 3 CLU Inhibits MMP-9 and MMP-2 Activity

Most MMPs are secreted as inactive pro-MMPs. The N-terminal propeptideblocks the catalytic domain by binding intramolecularly through thecysteine-zinc bridge between these two domains [Van Wart, 1990]. Enzymeactivation occurs upon cleavage or processing of the propeptide domain[Van Wart, 1990; Chen, 1993]. To determine whether MMP-9/CLU interactionaffects the cleavage process of the propeptide of MMP-9 and/orinterferes with enzymatic activity of the processed active enzyme, weperformed an in vitro MMP-9 inhibition assay, by adopting FRET assayusing a fluorescence-quenched peptide substrate (FIG. 3A). In thepresence of APMA the propeptide domain of pro-MMP is removed and thus itis activated in vitro [Visse, 2003]. APMA has also been shown to induceself-processing at a cryptic site at Ala³⁹⁸-Leu³⁹⁹ in the C-terminalregion of the MMP-9 [Murphy, 1995]. We incubated 1.5 pro-MMP-9 with PBS,CLU, SB-3CT, or BSA, and a fluorescence peptide substrate in thepresence or absence of APMA. SB-3CT is a potent chemical inhibitor ofgelatinases, MMP-2 and MMP-9, with Ki values in the sub-micromolar range[Kleifeld, 2001]. We monitored the change in fluorescence value duringthe incubation of the reactions up to 3 h, which reflects the enzymaticactivity of APMA-activated MMPs. We did not see any apparent change inthe fluorescence values' in the APMA-omitted reactions over theincubation time (data not shown), indicating that without APMA pro-MMP-9could not get activated. At 3 h post incubation, the activity differencewas read and reaction products were resolved by gelatin zymography. Theresults showed that CLU and SB-3CT inhibited the activity of MMP-9 by28% and 32%, respectively, compared with PBS and BSA. Three independentexperiments in triplicate each showed an average of 35% inhibition(p=0.002, student t-test) at 50 μg/ml of CLU (data not shown). When theprocessed enzyme products were resolved on the gel, three bands weredetected in the APMA-treated samples, pro-MMP-9, propeptide-cleavedMMP-9 (MMP-9), and MMP-9 with both propeptide and C-terminal regioncleaved (MMP-9-ΔC), whereas only pro-MMP-9 was detected in APMA-omittedsamples. The extent of MMP-9 processing among the four reactions withAPMA treatment was very similar, suggesting that CLU did not affect theenzyme processing by APMA. Thus, we suggest that CLU inhibit theenzymatic activity of MMP-9 without interfering with the APMA-inducedprocessing of pro-MMP-9. It has been known that CLU is present at 50-100μg/ml in the human normal serum [Viard, 1999; Jenne, 1991]. We testeddose-dependent inhibition of MMP-9 with CLU or SB-3CT (FIG. 3B). Theresult showed that CLU is a stronger MMP-9 inhibitor than a syntheticgelatinase inhibitor, SB-3CT. In order to test the specificity ofCLU/MMP-9 interaction, we also performed the inhibition assay with MMP2(FIG. 3C). Our results showed that CLU inhibited MMP-2 activity by 72%.The gel analysis indicated that the processing of MMP-2 was not alteredamong the different reactions, consistent with MMP-9 results. Comparisonof extents of MMP-2 or -9 inhibition by CLU (50 μg/ml or 0.63 μM, MW=˜80KDa) and SB-3CT (1 μM) in FIG. 3B suggests that CLU is a more potentinhibitor of both MMP-9 and MMP-2 than SB-3CT.

Example 4 Inhibition of MMPs without a Hemopexin-Like Domain at theC-Terminus, Including MMP-7

Most members of MMPs, except for MMP-7, MMP-23, and MMP-26, contain ahemopexin-like domain at the C-terminus of the enzyme, which modulatesthe processing and activity of the enzymes by serving as a bindingregion for regulatory or target proteins [Visse, 2003]. The initialbinding assay above utilized the full length of active form of MMP-9. Inorder to refine the region important for the MMP-9/CLU interaction, weused MMP-9 catalytic domain, which lacks the C-terminal regionencompassing the hemopexin-like domain (FIG. 4A). CLU inhibited MMP-9activity by 59%, indicating that terminal region of MMP-9 is notrequired for inhibition. To further confirm our results, we tested MMP-7which naturally lacks a C-terminal hemopexin-like domain (FIG. 4B). Ourresults showed that CLU inhibited APMA-activated MMP-7 by 17%, whileSB-3CT (160 μM) by 49%, and both results were statistically significant.Together, our results indicate that CLU can inhibit the enzymaticactivity of various members of MMPs, and does not depend on theC-terminal hemopexin-like domain for its function.

Example 5 CLU Enhances the Solubility of MMP-9 and MMP-2 in HydrophobicConditions

CLU interacts with various binding partners under differentphysiological conditions; however, the nature of their interaction ispoorly understood. In order to assess the nature of the MMP-9/CLUinteraction varying buffer conditions were investigated. In ourexperiments above (FIG. 3B), we used a low concentration of Brij 35(0.002%). For our first experiment here we analyzed on the solubility ofMMP2 and MMP-9 in three different nonionic detergents, Brij 35, NP-40,and N-octyl glucopyranoside (OG), at 0.04% in the presence or absence ofCLU (FIG. 5A). The soluble and insoluble fractions were separated, andwere resolved on the gelatin zymography. Our results indicated that Brij35 and NP-40 showed no effect on the solubility of both MMP-2 and MMP-9regardless of the presence of CLU. Interestingly, in the presence of OGthe solubility of both enzymes decreased; however, OG failed to reducetheir solubility in the presence of CLU. These results suggest apossibility that CLU may render pro-MMP-2 and MMP-9 soluble in certainhydrophobic conditions. Next, we performed the inhibition assay usingpro-MMP-2 in the buffer containing 0.04% Brij 35 or NP-40. As shown inFIG. 5B, MMP-2 activity was inhibited by CLU in both buffers to theextent similar to that in the lower detergent concentration as shown inFIG. 3B. Furthermore, the level of inhibition shown by SB-3CT and BSAalso revealed the similarity with that of a lower concentration ofdetergent, suggesting that MMP-2/CLU interaction may not be sensitive tothe types or concentration of detergents. However, when the similarexperiments were performed with pro-MMP-9 or APMA-activated MMP-9, thehigh concentration of Brij 35 or NP-40 did show little MMP-9 inhibitionby CLU (FIG. 5C). These results indicate that high concentration ofnonionic detergents may affect the ability of CLU to inhibit the MMP-9but not MMP-2 activity.

Example 6 CLU Binds to Pro-MMP-9 with Higher Affinity than ProcessedMMP-9

Since different detergent concentrations affected the extent ofinhibition of MMP-9 activity by CLU, we tested whether detergents mayinfluence the binding affinity of CLU for MMP-9. To test thispossibility, CLU pull down assay was performed. Pro-MMP-9, MMP-9, andMMP-9-ΔC were mixed and incubated with CLU-His in two differentconcentrations of Brij 35. MMP-9 enzymes bound to CLU were isolatedusing anti-His tag antibody, and subjected to gelatin zymography toassess the relative amounts of different forms of MMP-9 (FIG. 6A). Inthe input lanes on the gel, the relative amount of pro-MMP9 was muchlower than the other two forms of MMP-9. In contrast, lanes containingMMP-9 bound with CLU showed enrichment predominantly of pro-MMP-9 formcompared with the densities of the other two forms. Analysis of thedensities of individual bands using Image J software suggested thatpro-MMP-9 may have at least 10 times stronger affinity for CLU than theother forms of MMP-9. The levels of pro-MMP-9 have little differencebetween low and high detergent concentrations, indicating the affinityis not sensitive to the detergent concentration. However, the amounts ofMMP-9 and MMP-9-ΔC in the high Brij35 concentration were much lower thanthose in the low concentration and similar to those in beads control,suggesting that MMP-9 and MMP-9-ΔC do not bind to CLU at the high Brij35 concentration, consistent with the failure of CLU inhibition of MMP-9activity in that condition.

The above results suggest that the propeptide domain is essential forhigh affinity binding of CLU with pro-MMP-9. To confirm this further, wecleaved pro-MMP-9 bound to CLU on beads with APMA, and separatedsupernatant and bead fractions (FIG. 6B). We reasoned that if CLU bindsto the propeptide domain, the processed active form of MMP-9 should beenriched in supernatant fraction (S), and, if it binds to the other partof the enzyme, the processed enzyme should be in the beads fraction (B).When the samples were resolved on the gel, pro-MMP-9 was equallydistributed into S and B fractions in the untreated reaction, suggestingthe release of portion of enzymes from the beads into solution duringthe incubation. However, in APMA-treated reaction, a large fraction ofthe processed MMP-9 was detected only in the S fraction, indicating agreater affinity at the propeptide domain of MMP-9, with a loweraffinity binding sites located elsewhere.

Example 7 CLU Expression in the Human Corneal Epithelial Cells

To determine how human corneal epithelial cells express and secrete theCLU protein, we used an immortalized human corneal epithelial cell line(HCLE) that was kindly provided by Dr. Ilene Gipson (HarvardUniversity).

We isolated total RNA from the HCLE cells as well as from human cornealepithelial tissue and human periodontal ligament stem cells for thesynthesis of cDNA. This cDNA was then used for PCR amplification withthree different sets of primers designed previously in order to detectthe three different isoforms of CLU transcripts (Andersen et al., 2007)(FIG. 8A), presumably transcribed by three different transcriptionalinitiation sites (Cochrane et al., 2007). Form 1 (isoform 1) is for thetranscript of NM_(—)001831, and form 2 (isoform2), for NM_(—)203339 inthe GeneBank DNA data base.

While all three transcript isoforms produce the secreted form of CLU,only transcript isoform 1 has a potential to produce the transcriptslacking exon 2 which can be translated into the nuclear CLU protein(Leskov et al., 2003). Our results indicate that HCLEs and cornealepithelial cells express isoform 1 as a dominant transcript. HCLE cells,however, also express transcript isoforms 2 and 3 while human cornealepithelial cells only express transcript isoform 1. This difference maybe due to the difference between cultured cells and fresh tissue.Culturing of human corneal epithelial cells may induce the expression oftranscript isoforms 2 and 3. There was no significant differenceobserved between the densities of isoforms 1 and 2 in the periodontalstem cells. Overexpression of isoform 2 was shown to render certainprostate cells more resistant to Fas-mediated cell death (Miyake et al.,2001).

FIG. 8B shows that CLU is secreted into culture media from the cornealcells. Briefly, the media from the HCLE cell culture was subject toWestern blotting with an anti-CLU antibody (Santa Cruz Biotech.,sc-6419). Lane 1 contains conditioned medium (K-sfm) from the cellculture; lane 2 has regular medium (K-sfm plus BPE and EGF) from theculture; and lane 3 includes fresh regular medium as a control. Thearrow indicates the position of the β-subunit of CLU with the expectedsize of ˜40 RD.

Example 8 CLU Processing is Influenced by Growth Conditions

To test if there are any changes in CLU expression and processing inresponse to the different growth conditions, we altered cell cultureconditions. We grew the HCLE cells to a subconfluent cell density orkept them at a confluent cell density so that they underwent contactinhibition in the regular K-sfm medium. We also induced stratificationof the corneal cells by replacing the regular medium with DMEM:F12medium for the confluent cells (FIG. 9) (Gipson et al., 2003). Thedistribution of the intracellular CLU protein undergoing processing wasnot influenced by the cell density (subconfluent vs. confluent) but wasaffected by different media (K-sfm vs. DMEM:F12), which showed that CLUintermediate protein bands increased or decreased in the stratified cellculture. The data suggests that the environmental factors affecting thecorneal cells may alter the production or processing of mature CLUprotein in cells.

Accumulating evidence supports the concept that MMP-9 isdisease-promoting whereas CLU is preventive in situations involvinginflammatory processes. We found that MMP-9 and CLU interact with eachother inside and outside cells. We showed that co-expression of MMP-9and CLU delays the intracellular processing of the CLU proteins, andthat CLU inhibits MMP-9 activity in vitro. The current observationssuggest that MMP-9 and CLU may influence each other's regulation andactivity in an antagonistic manner, so there may be opposing roles forMMP-9 and CLU in the progression and development of inflammatory diseasesuch as dry eye.

Our experiments show that CLU is expressed by corneal epithelial cellsand binds to and inhibits MMPs, which are known to be involved in woundprogression and symptoms of dry eye disease. Therefore, CLU can be usedas a biomarker for the disease progression as well as a therapeuticagent for the prevention and treatment of dry eye and promotion of woundhealing.

Example 9 Clusterin Inhibits the Induction of MMP-9 by TNF-Alpha fromHuman Corneal Limbal Epithelial (HCLE) Cells

TNF (tumor necrosis factor)-alpha is one of key cytokines involved ininflammatory events. MMP (matrix metalloproteinase)-9 also plays a rolein the pathological inflammatory process. TNF-alpha is known to induceMMP-9 from certain cell types such as HCLE cells.

HCLE cells were grown to be confluent in KSFM media and then replenishedwith DMAM/F12 media to stratify cells for 7 days. To these cells inserum-free DMEM/F12 media, clusterin (50 ug/ml), bovine serum albumin(BSA, 50 ug/ml), and TNF-alpha (10 ng/ml), individually or incombination, were treated for 24 hours. The same volume of supernatantsof the cell cultures treated were collected to resolve on SDS/PAGE gelcontaining gelatin in order to perform gelatin zymography to visualizethe presence of MMP-9 secreted from the cells. When compared withuntreated (Un), MMP-9 density increased upon TNF treatment (TNF),indicating that TNF induced MMP-9 secretion from the cells. However,when TNF was treated with CLU (TNF+CLU) did not increase MMP-9 secretionany longer, suggesting that CLU inhibited TNF-induced MMP-9 secretion.Therefore, Clusterin inhibits the induction of MMP-9 from human corneallimbal epithelial (HCLE) cells. (See FIG. 10)

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It should be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It should be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not, be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All references cited herein, including but not limited to patents,patent applications, and non-patent literature, are hereby incorporatedby reference herein in their entirety.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of themethod and compositions described herein. Such equivalents are intendedto be encompassed by the following claims.

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What is claimed is:
 1. A method of treating dry eye disease comprising:administering to a patient in need thereof an effective amount of apharmaceutical composition comprising an isolated clusterin or anisolated fragment of clusterin, wherein said clusterin or said fragmentof clusterin binds and reduces the activity of at least one of MMP-9,MMP-2, or MMP-7.
 2. The method of claim 1, wherein the pharmaceuticalcomposition comprises secreted clusterin.
 3. The method of claim 1,wherein the administration is topical.
 4. The method of claim 1, whereinthe pharmaceutical composition further comprises a liquid carrier, andadministration is by contacting the pharmaceutical composition to thesurface of an eye of the patient.
 5. The method of claim 1, wherein thepharmaceutical composition further comprises a carrier.
 6. The method ofclaim 1, wherein the carrier is a sterile solution.